Gel extraction - CHARGE syndrome: candidate genes and pathogenesis

2.2 Methods

2.2.11 Gel extraction

The QIAquick Gel Extraction Kit (Qiagen) is used to extract and purify DNA (70 bp-10 kb) from agarose gels in TAE or TBE buffer according to the manufacturer’s instructions. All centrifugation steps were carried out at 13,000 rpm at room temperature. The desired DNA fragment was excised from the agarose gel using a clean scalpel. If possible, extra agarose was removed before the gel slice was scaled in a colourless tube. To 1 volume of gel (1 mg is equivalent to 100 µl) 3 volumes of Buffer QG were added to the gel slice. In case of > 2 % agarose gels, 6 volumes Buffer QG were needed. Next, the tube with the gel slice was incubated 10 minutes at 50 °C until the gel slice was completely dissolved. Every 2-3 minutes the tubes were vortexed during incubation to support dissolution. Since the adsorption of DNA to the QIAquick membrane is efficient only at pH ≤ 7.5, it is necessary that the colour of the mixture stays yellow indicating the required pH. In case the mixture is orange or violet after the gel has dissolved, 10 µl of 3 M sodium acetate (pH 5) had to be added. To DNA fragments < 500 bp and > 4 kb 1 gel volume of isopropanol was added which increases the yield of DNA fragments. Next, a QIAquick spin column was placed in the provided 2 ml collection tube before the sample was applied to the column to bind DNA. The probes were centrifuged for 1 minute. The column reservoir is 800 µl. For sample volumes > 800 µl, the column was simply loaded and centrifuged again. The flow-through was discarded and the column

placed back in the same collection tube. 500 µl Buffer QG was added to the column and centrifuged for 1 minute. The flow-throw was discarded and 750 µl of Buffer PE was added to the column for washing. After 2-5 minutes incubation, the column was centrifuged for 1 minute. The flow-through was removed and the column centrifuged again for 1 minute to completely remove the ethanol from the Buffer PE. To elute the DNA, the column was placed into a new 1.5 ml e-cup and 30-50 µl ddH2O was added to the center of the QIAquick membrane. After 1 minute incubation the column was centrifuged for 1 minute. The DNA samples were stored at -20 °C 2.2.12 Mouse preparation

One-to-one matings of heterozygous Whirligig (Chd7^Whi/+) females and males were set up. To determine the age of the embryos, a daily check for a vaginal plug (VP check) was performed on female Whirligig mice. The day when the copulation plug was observed, was counted as embryonic stage 0.5 (E0.5). At the required day the pregnant Whirligig mice were sacrificed by cervical dislocation and the uterus was removed. The embryos were dissected from the yolk sacs and washed with PBS. To determine the gender of the embryos, a tail biopsy was performed. For protein and RNA isolation the embryos were flash frozen on liquid nitrogen and stored at -80 °C.

For paraffin sections the embryos were fixed overnight at 4 °C in 4 % paraformaldehyde.

2.2.13 Fixation and dehydration of embryos for paraffin embedding

Fixation and dehydration of mouse embryos have been carried out according to Luxan et al. (2013) with some modifications, such as additional incubation steps and longer incubation times. Mouse embryos isolated from the uterus were fixated overnight in 4 % paraformaldehyde in PBS at 4 °C. The next day embryos were washed for 10 minutes in PBS. If not indicated differently, all steps were carried out at room temperature. By an increasing ethanol row (50 %, 70 %, 80 %, 90 % ethanol/PBS and 100 % ethanol) embryos were dehydrated. In contrast to the procedure performed by Luxan et al. (2013) an additional 50 % ethanol/PBS incubation step was carried out and each concentration step was performed twice for 1 hour. After the dehydration, embryos were incubated overnight instead of 30 minutes in 100 % xylene. Additionally, embryos were incubated in 50 %

paraffin/xylene for 2-6 hours at 65 °C. Embryos were transferred into 100 % paraffin and incubated for 2 days instead of 3 hours at 65 °C. During that time the paraffin was exchanged at least six times. The embryos were ready to be placed into forms to make paraffin blocks for the preparation of sections.

2.2.14 Preparation of paraffin sections of mouse embryos

To prepare paraffin sections, the microtome (JUNG RM 2035, Leica) were used.

First, the paraffin block with the fixed embryo was cut to the size needed and clamped onto the microtome. Sections of 6-8 µm thickness were made which were carefully transferred to the surface of water (58 °C) from where they were placed on slides (Thermo Scientific). After the sections were dried, they were stored at 4 °C or room temperature until needed.

2.2.15 Microarray and data analysis

Microarrays are used to measure the expression of a large number of genes (Schena et al., 1995). In this work the microarrays were performed by the TAL in Goettingen as described by Schulz et al. (2014b) using the "Low RNA Input linear Amplification Kit Plus, One Color" protocol (Agilent Technologies, Inc. 2007; Cat. N°: 5188-5339) and the RNA Spike-In Kit for One color (Agilent Technologies, Inc. 2007; Cat. N°:

5188-5282) following the manufacturer's instructions. For the analysis of global gene expression the Mice 4 x 44 K design array from Agilent Technologies (G4846A) was used following the manufacturer’s standard protocol. 200 ng of total RNA was used for reverse transcription into cDNA following the company’s recommendation. The in vitro transcription was done according to the manufacturer’s instructions. To determine the quantity and efficiency of the amplified cRNA that was labeled, the NanoDrop ND-1000 UV-VIS Spectrophotometer version 3.2.1 was used. The hybridisation procedure took place in the Hybridisation Oven (Agilent). Probes were incubated for 17 hours at 10 rpm at 65 °C. The following washing and staining were performed according to the manufacturer’s standard protocol. All together the microarray data were generated according to the Minimum Information About a Microarray Experiment (MIAME) guidelines (compare NCBI's Gene Expression Omnibus (GSE46591). The Agilent DNA microarray scanner (G2505B) was used at 5 micron resolution to detect the Cy3 intensities by one-color scanning. Next, the

scanned image files were screened for artefacts and further analysed conforming to Opitz et al. (2010). The Feature Extraction Software Version was changed to 10.7.3.1. DAVID was used to find over-represented functions (Huang da et al., 2009b, a)

2.2.16 Cell biological methods

2.2.16.1 Culturing of eukaryotic cells

In this work human HeLa cells derived from cervical cancer cells were used (Scherer et al., 1953). The adherent cells were cultured in 25 cm² and 75 cm² culture flasks (Sarstedt) in 5 ml and 10 ml medium, respectively. The medium consists of Dulbecco’s modified Eagle’s media (DMEM) (PAN BIOTECH), 10 % foetal calf serum (PAN BIOTECH) and 1 % penicillin/streptomycin (PAN BIOTECH). Cells were cultured at 37 °C at a humidity of 98 % and CO₂ content of 5 %. Every two days the medium was exchanged.

To split cells, TrypLE Express (Invitrogen) was used to detach cells from the culture flask. First, cells cultured in a 25 cm² flask were washed once or twice with 5 ml DPBS. DPBS was removed completely before adding 1.5 ml TrypLE Express. Cells were incubated at 37 °C for 3 minutes. Then, 3.5 ml medium was added and cells were resuspended and transferred to a 13 ml centrifugation tube and centrifuged at 1,000 rpm for 5 minutes at room temperature. Supernatant was removed and the cell pellet was washed by resuspending in 5 ml DPBS. Tubes were centrifuged again at 1,000 rpm for 5 minutes at room temperature. The supernatant was discarded and the cell pellet was resuspended in medium. A few drops were added to a new 25 cm² culturing flask with 5 ml fresh medium. Cells were equally distributed and incubated at 37 °C.

2.2.16.2 Cryopreservation and revitalisation of eukaryotic cells

For a permanently storage, cells were cultured in 75 cm² flask (Sarstedt) until they reached a confluence of 80 %. They were washed with DPBS and then trypsinised (2.2.16.1). Cells were resuspended in 1.5 ml DMEM, and 1.5 ml of the Cryo-medium consisting of 50 % FKS, 30 % DMEM and 20 % DMSO was added. Cell mixture was transferred in Cryo-tubes and temporarily incubated at -20 °C. Then, cells were kept at -80 °C or liquid nitrogen. For revitalisation, cells were quickly unfrozen at

room temperature and resuspended in 9 ml prewarmed culturing medium. Cells were pelleted twice, washed with culturing medium and cultured in a 25 cm² flask (Sarstedt) with fresh medium (2.2.16.1). After 24 hours the medium was exchanged.

2.2.16.3 Transfection of eukaryotic cells with plasmids

Transfection is the introduction of nucleic acids (DNA or RNA) into cells. In this work HeLa cells were transfected with plasmids containing either a complete gene sequence or certain segments of a gene sequence to overexpress the appropriate DNA sequence in vivo. As transfection reagent lipofectamine 2000 (Invitrogen) was used according to the manufacturer’s instructions. Lipofectamine reagent forms cationic liposomes in an aqueous environment which entrap the negatively charged transfection material by forming complexes (Dalby et al., 2004). The cationic lipid molecules are formulated with a neutral lipid that ensures the introduction of the transfection material into the cells (Dalby et al., 2004). HeLa cells were cultured 24 hours before transfection in 75 cm² flasks (Sarstedt) so they have a confluence of 70-90 % at the time of transfection. 30 µg of plasmid DNA was mixed with 1.875 ml Opti MEM (Invitrogen) and incubated for 5 minutes at room temperature. 75 µl lipofectamine 2000 was also mixed with 1.875 ml Opti MEM and incubated for 5 minutes at room temperature. The plasmid DNA and the the lipofectamine mixture were combined and incubated for 25 minutes at room temperature. In the meanwhile cells were washed with DPBS and 9 ml DMEM with 1 x NEAA (Gibco) was added.

The transfection mixture was added to the cells trop by trop and then incubated at 37 °C. After 4-5 hours, 9 ml DMEM with 20 % FKS and 1 x NEAA was added to the medium. After transfection, cells were incubated at 37 °C for 24 hours total.

2.2.17 Protein chemical methods

2.2.17.1 Protein isolation from HeLa cells

Nuclear proteins from HeLa cells were specifically isolated using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific) according to the manufacturer’s instruction. First, the medium were removed and cells were washed twice with DPBS and then harvested using TrypLE Express (Invitrogen). To neutralise the trypsin, DMEM medium was added instead of DPBS and the cell suspension was centrifuged at 500 x g for 5 minutes. The supernatant was removed

and the cell pellet washed by suspending in DPBS. The cell suspension was centrifuged again at 500 x g for 5 minutes. The supernatant was discarded and the cell pellet suspended in 1.5 ml DPBS and then transferred into a 1.5 ml e-cup.

Following centrifugation steps were performed at 4 °C, cell samples and extracts were kept on ice. Next, the cells were centrifuged at 500 x g for 2-3 minutes. The supernatant was carefully removed to leave the cell pellet as dry as possible.

Depending on the cell volume the reagent volume was determined as shown below.

Table 36: Reagent volumes for different packed cell volume

Packed Cell Volume (µl) CER I (µl) CER II (µl) NER (µl)

10 100 5.5 50

20 200 11 100

50 500 27.5 250

100 1,000 55 500

Right before using CER I and NER protease inhibitors (100 x stock) were added to maintain extract integrity and function. First, ice-cold CER I was added to the cell pellet which was then fully suspended by vortexing on the highest setting for 15 seconds. After 10 minutes incubation, the appropriate amount on ice-cold CER II was added. The tube was vortexed for 5 seconds on the highest setting, then incubated for 10 minutes and vortexed again for 5 seconds on the highest setting.

Samples were centrifuged for 5 minutes at maximum speed (~ 16,000 x g). The supernatant consisting of the cytoplasmic extract was transferred to a new prechilled tube. To the pellet which contains the nuclei, the appropriate amount on ice-cold NER was added. For a total of 40 minutes the tube was vortexed every 10 minutes for 15 seconds on the highest setting and then centrifuged for 10 minutes at maximum speed (~ 16,000 x g). The supernatant which contains the nuclear extract was transferred to a new prechilled tube. The protein concentration was determined by Synergy Mx spectrophotometer (BioTek). Extracts were aliquoted and stored at

80 °C.

2.2.17.2 Measurement of protein concentration

After the principle according to Bradford (1976), the concentration of proteins was measured. Therefore, Roti^®-Nanoquant (Roth) was employed as described by Witt

(2013). This solution contains Coomassie Brilliant Blue that binds under acidic conditions to proteins which leads to a conversion of the red form to the blue form of the dye causing a shift in the absorption spectrum maximum from 365 nm to 595 nm (Bradford, 1976). A standard protein, namely BSA (0-6.7 µg BSA/ml), was used for calibration. Based on the standard curve the protein concentration could be extrapolated. A triple measurement of each sample was performed and the average of the values was determined. First, a 1:100 dilution of the desired protein extract was prepared. 50 µl of the diluted protein extract and 50 µl of ddH2O as a reference were put in a 96 well plate and 200 µl of the Roti^®-Nanoquant solution was added. After 5 minute incubation at room temperature, the absorption was measured at 590 and 450 nm by Synergy Mx spectrophotometer.

2.2.17.3 Co-IP

The Co-IP is a technique used to analyse protein-protein interactions. With an antibody a known protein is targeted which is believed to be a member of a protein complex, the protein antigen is precipitated as well as proteins that are bound to it.

By western blot analysis and further protein detection methods it might be possible to identify unknown complex members. For Co-IP nuclear cell extract was used and the immunoprecipitation of the target protein was carried out using the Immunoprecipitation Kit (Protein G) from Roche according to the manufacturer’s instructions with modifications regarding incubation times, number of washing steps and during centrifugation steps. Depending on the amount of nuclear cell extract, an appropriate amount of the specific antibody was added and the sample incubated on a rotating platform overnight at 4 °C (Roche protocol: 1 hour). The next day 45 µl of the protein G was added to a new tube and centrifuged for 1 minute at 13,000 rpm at room temperature (Roche protocol: 50 µl). The supernatant was removed and the overnight protein solution was added to the remaining protein G. Next, the mixture was incubated on a rotating platform for 2 hours at 4 °C (Roche protocol: 3 hours).

Complexes were collected by 3 minutes centrifugation at 3,000 rpm at 4 °C (Roche protocol: 12,000 x g for 20 sec). After the supernatant was removed, the beads were washed using 500 µl of the lysis buffer (Roche protocol: 2 times 1 ml lysis buffer and 20 minutes incubation). The samples were centrifuged again for 3 minutes at 3,000 rpm at 4 °C (Roche protocol: 12,000 x g for 20 sec). The beads were resuspended in 1 ml wash buffer 2 and centrifuged under the same conditions as

before (Roche protocol: centrifugation at 12,000 x g for 20 sec after 20 minutes incubation). After the supernatant was removed, a final washing step using 1 ml wash buffer 3 was performed (Roche protocol: 2 times 1 ml wash buffer 3 and 20 minutes incubation). The following preparation to load the sample on an SDS gel was performed differently. The supernatant was removed after centrifugation and 18 µl DPBS (PAN), 18 µl LDS (Invitrogen) and 4 µl 1M DTT were added to the beads. To denaturise proteins and separate them from the protein G beads, the mixture was incubated for 5 minutes at 95 °C. Beads were centrifuged down and the supernatant was loaded on an SDS gel and further analysed by western blot (2.2.17.5).

2.2.17.4 SDS polyacrylamide gel electrophoresis

By SDS polyacrylamide gel electrophoresis (SDS PAGE) proteins are denaturised and can be separated according to their molecular weight in a polyacrylamide gel by an electric field (Shapiro et al., 1967; Laemmli, 1970). In this work 4-12 % Bis-Tris Gels (Invitrogen) and 3-8 % Tris-Acetate Gels 3-8 % Tris-Acetate Gels (Invitrogen) were used for protein separation (2.2.17.5).

2.2.17.5 Western blot

The western blot is an analytical technique and this method was used to transfer proteins from an SDS gel to a nitrocellulose membrane where the proteins can be detected by different reactions (Towbin et al., 1979; Towbin and Gordon, 1984;

Towbin et al., 1992).

Protein extracts used for Co-IP were incubated with 18 µl DPBS (PAN), 18 µl LDS (Invitrogen) and 4 µl 1M DTT for 5 minutes at 95 °C. After spinning down the Protein-G-agarose beads, 30 µl of the supernatant which contains the precipitated protein along with other proteins bound to it in a denaturised form was loaded on an SDS gel. Two different transfer methods were used in this work.

a) semidry

With this transfer variant proteins < 100 kDa were transferred from an SDS gel to a membrane. Using MES SDS Running Buffer (Invitrogen) and a 4-12 % Bis-Tris Gel (Invitrogen), proteins were separated according to their molecular weight. For approximately 2.5-3 hours the gel was running at 100 V. The protein transfer was

performed according to Wieczerzak (2012). The blotting condition was changed to 220 mA instead of 230 mA. 4 Whatman Papers which were soaked in transfer buffer were placed on the Turboblotter^TM (Schleicher & Schuell). A nitrocellulose membrane which was wetted in ddH2O and transfer buffer was put on top of it followed by the SDS gel and another 4 prewetted Whatman Papers. Approximately 50 ml transfer buffer were needed. The blotting was performed at constant 150 mA for 15 minutes followed by 45 minutes at 220 mA.

b) wet blot

This transfer method is appropriate for larger proteins (> 100 kDa). First proteins were separated according to their molecular weight using gel electrophoresis.

Therefore, Acetate SDS Running Buffer (Invitrogen) and 3-8 % Tris-Acetate Gels (Invitrogen) were used. The running time of the gel amounted 3-3.5 hours at 100 V.

To prevent heating of the buffer, the gel chamber was placed in an ice box. Once the proteins were separated, the “gel-sandwich” was assembled in transfer buffer to avoid bubbles according to BIO-RAD (n.d.). On the grey coloured side of the cassette (Bio-Rad) a foam pad was placed followed by 2 Whatman Papers, a nitrocellulose membrane and the SDS gel. Finally, 2 Whatman Papers and another foam pad were placed on top. The cassette was closed and placed vertically between parallel electrodes in the buffer tank (Bio-Rad) containing a frozen blue cooling unit and the transfer buffer (Tab. 13). To avoid the formation of an ion gradient and to insure the temperature homogeneity, a magnetic stir bar was placed in the buffer tank which was put in a box filled with ice on a magnetic stirrer. For the blotting at 4 °C, the voltage was set on 60 V constant for 2.5 hours.

2.2.17.6 Protein detection using antibodies

Once the protein transfer from the SDS gel to the membrane was completed, the membrane was transferred into a falcon tube (CellSTAR) and unspecific binding sites were blocked by incubating the membrane in TBST with 5 % milk or 5 % BSA for 1-2 hours at room temperature. During incubation steps membranes were placed on a rolling platform. The membrane was then incubated overnight at 4 °C with the appropriate primary antibody. Antibodies were diluted 1:1,000 to 1:2,000 in TBST with 2 % milk or 2 % BSA. Next day the membrane was washed three times for 10 minutes in 20 ml TBST with 2 % milk or TBST if BSA was used. The membrane

was then incubated for 2 hours at room temperature or at 4 °C with a secondary antibody conjugated to horseradish peroxidase (HRP). The antibodies were 1:7,000 to 1:10,000 diluted in TBST with 2 % milk (incubation at room temperature) or 2 % BSA (incubation at 4 °C). After that, the membrane was washed three times for 10 minutes in TBST with 2 % milk and once with TBST for 5 minutes or three times for 10 minutes with TBST at room temperature. To visualise proteins, SuperSignal^TM West Pico Chemiluminescent Substrate (Thermo Scientific) was used according to the company’s instructions. To prepare the substrate working solution, the two substrate components were mixed in a 1:1 ratio (light sensitive). The membrane was placed between a clear plastic wrap and incubated 5 minutes in the dark with SuperSignal West Pico Substrate Working Solution. HRP produces a detectable light signal in the presence of a substrate which can be either detect on X-ray films or by the detecting system FlourChem^® Q (Alpha Innotech).

2.2.18 Direct yeast two-hybrid

The direct Y2H assay is used to identify and investigate protein-protein interactions in vivo using yeast cells. Two proteins of interest are expressed separately. The protein functioning as a bait protein is fused to the Gal4 DNA-binding domain (e.g.

pGBKT7 plasmid) while the other one functions as the prey protein which is fused to the Gal4 transcriptional activation domain (e.g. pGADT7 plasmid) (Fields and Song, 1989). Only if a cell contains both proteins that interact with each other, the Gal4

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